Solid-core ring-magnet

ABSTRACT

A solid-core ring-magnet having one or more cavities is provided. The magnet can have an overall cylindrical shape or a rectangular-prism shape. In either case, a portion of cavity walls of the magnet are ring shaped, causing the magnetic field lines to emanate from the magnet in the shape of a ring. The diameter of the ring shaped cavities can be constant throughout, constant through a portion of the cavity, variant throughout, or variant through a portion of the cavity. The cavities open to the end of the magnet, and terminate toward the core of the magnet. Also provided are systems and kits having solid-core ring-magnets. Methods of purifying a macromolecule using the solid-core ring-magnets are also provided.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/515,256, entitled “SOLID-CORE RING-MAGNET” by Olaf Stelling, filedOct. 15, 2014. The entire teachings of the above application areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Isolation of macromolecules (e.g., nucleic acids, such as DNA or RNA,and proteins such as antibodies) is required before they can be used inmany applications. For example, sequencing of nucleic acids andrestriction digestion of nucleic acids requires or at least benefitsfrom their purification. Nucleic acids can be purified via a variety ofmethods, including the traditional phenol-chloroform extraction. Arelatively modern method of purifying nucleic acids makes use ofmagnetic beads. In this approach, magnetic beads are coated with asubstance to which nucleic acids have affinity under certain conditions,and from which nucleic acids can be separated under differentconditions. Employment of magnetic beads in this manner can eliminate aneed for centrifugation steps or vacuum filtration steps, can speed upthe process, can increase the yields of recovery, and can allow recoveryof nucleic acids directly from an initial sample. Centrifugation andvacuum filtration have traditionally been difficult to automate.Magnetic beads can similarly be used for macromolecules other thannucleic acids; they can be used for proteins and complexes of two ormore macromolecules.

Use of magnetic beads, for example while preparing samples for DNAsequencing, suffers from requiring collection of DNA from a relativelylarge volume, from the recovered DNA being diluted in the solution, andfrom the form of the recovery vessel being restricted based on thepurification setup used. A need exists to improve time sensitive,high-throughput applications with the use of magnetic beads. Therefore,there is a need for improved apparatuses and methods that can enablepurification of macromolecules in more concentrated solutions and withina wider variety of vessels.

SUMMARY OF THE INVENTION

Macromolecules, such as nucleic acids, especially those of high qualityand purity, can be obtained via a variety of methods. In one method,complexes are formed between macromolecules and magnetic beads, and themagnetic beads are separated from a mixture, essentially purifying themacromolecules after their “un-complexation” from the beads throughchanges in conditions. In an embodiment, the complex between themacromolecules and magnetic beads remains in the vessel in the form of aring and most of the solution is removed, leaving a high concentrationof complex in the vessel.

In an embodiment, the present invention includes a magnet that can beused to isolate/purify macromolecules from a mixture. The mixture, asdefined herein, is any aqueous solution that has at least themacromolecule in addition to the solvent. As an example, it can beextracellular matrix. The macromolecules, as defined here, encompassnucleic acids such as DNA or RNA, or proteins such as antibodies. Themagnet, in particular, can be used to isolate macromolecules by makingthem adhere to magnetic beads, after which they can be separated fromthe mixture. In particular, through changes in the chemical environmentmacromolecules are made to adhere to the magnetic beads to form acomplex. The magnet is then used to attract the complex, and pull themout of solution. In particular, the magnet of the present inventioncauses the complex to form a ring of bead complexes within the vessel.The solution can then be removed leaving behind the magnetic beads withthe macromolecules adhered thereto.

The magnet encompassed by the present invention has a top surface, abottom surface, a solid core, and one or more cavities. Each cavitystarts at a surface and goes toward the center of the magnet, but doesnot reach the other side thereby leaving a solid core intact. In otherwords, no tunnel from the top to bottom surfaces is formed and themagnet retains a solid core. The magnet is surrounded by a side wall onits sides not covered by the surfaces (the top and bottom surfaces).

In an embodiment, the magnet has an overall cylindrical shape. Inanother embodiment, the magnet is shaped like a rectangular prism. Ineach of these, the cavities are formed. In embodiments, the cavities canhave a ““U” shape, “V” shape or other irregular shape so long as it canreceive the vessel, as described herein. In a particular embodiment, thecavity wall of the inventive magnet has at least a top portion that isring-shaped, and other portions can be, for example, conical shaped. Thecavities are defined by their cavity walls. The cavity wall can includea base surface, which is the innermost part of the cavity wall thatterminates the cavity. The cavity walls can have a constant diameter, orthey can have varying diameters. In an embodiment, the base surface canbe conically shaped; thus, it might have progressively decreasing radiitoward the terminus of the cavity. The cavities receive vessels (e.g.,Eppendorf tubes, wells of a microplate) which hold a solution. When thevessel is placed in the cavity of the inventive magnet, the volume ofthe portion of the solution that falls inside or within the cavity andup to the macromolecule/bead ring, in an embodiment, is between about 5and about 30 micro-liters (e.g., between about 5 and about 25, 20, 15,and 10 micro-liters). In another embodiment, the volume of the cavityitself is between about 5 and about 45 micro-liters (e.g., between about5 and about 40, 35, 30, 25, 20, 15 micro-liters.

In another embodiment, a system for isolating macromolecules isdisclosed. In addition to the magnet, the system can include a vesselfor holding a mixture that includes a macromolecule (e.g., DNA). Thesame types of magnets as encompassed by other embodiments can beincluded as part of the system as well.

Also disclosed are methods of purifying macromolecules from a liquidsample that contains a mixture. The methods, in an embodiment, includesteps of collecting the liquid in a vessel, adding magnetic beads to thesample, and separating the magnetic bead-macromolecule complex from thesample by placing the vessel in a cavity of a magnet. After these steps,the macromolecule can be eluted from the magnetic beads.

In an embodiment, the present invention includes a kit. The kit cancomprise a magnet, as described previously, and a vessel for holdingliquid samples. In an embodiment, the vessel can be placed into a cavityof a magnet, and a volume of 5 to 35 micro-liters (e.g., between 5 and35, 5 and 30, 5 and 25, 5 and 20, 5 and 15, 5 and 10 micro-liters) ofsample would remain in the portion of the vessel that is within themagnet and up to the band. Magnetic beads can also be added as part ofthe kit in some embodiments.

Additionally disclosed are magnet plate systems for isolatingmacromolecules. The systems include at least one magnet as well as a topplate, a support plate, and a base plate. One or more springs woundaround one or more shoulder posts can also be included as part of themagnet plate systems. The top plate can include a plurality of magnetreceivers, and it can accommodate either cylindrical shaped magnets orblock shaped magnets.

There are many advantages provided by the disclosed systems. Betteryields of recovered macromolecules, faster recoveries, higherconcentrations, and higher purities of recovered macromolecules areattainable as compared to magnets and systems previously available.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like parts are referred to by thesame reference characters across different views. The drawings are notnecessarily to scale, emphasis instead being placed on illustrating theprinciples of the invention.

FIG. 1A is a schematic of a perspective view of a solid-core ring-magnethaving a cylindrical shape and cylindrical/conical-shaped cavities.

FIG. 1B is a schematic of a perspective view of a solid-core ring-magnethaving multiple cylindrical/conical-shaped cavities and an overallrectangular-prism shape.

FIG. 2A is a schematic of a top view of a solid-core ring-magnet havinga cylindrical shape.

FIG. 2B is a schematic of a top view of a solid-core ring-magnet havingmultiple cylindrical/conical-shaped cavities and an overallrectangular-prism shape.

FIG. 3A is a schematic of a cut-out side view, as defined in FIG. 2A, ofa solid-core ring-magnet having a cylindrical shape.

FIG. 3B is a schematic of a long-side view of a solid-core ring-magnethaving multiple cylindrical/conical-shaped cavities and an overallrectangular-prism shape.

FIG. 3C is a schematic of a short-side view of a solid-core ring-magnethaving multiple cylindrical/conical-shaped cavities and an overallrectangular-prism shape.

FIG. 4A is a schematic of a side view of a solid-core ring-magnet havinga cylindrical shape, further showing two cavities and a V-shaped vesselfor holding a reaction mixture of magnetic beads and macromolecules. Thering of complex between the magnetic beads and macromolecules just abovethe top of the magnet is shown.

FIG. 4B is a schematic of a side view of a standard ring magnet havingone full-length channel, and a V-shaped vessel for reaction mixture ofmagnetic beads and macromolecules. The ring of complex between themagnetic beads and macromolecules just above the top of the magnet isshown.

FIG. 4C is a schematic of a side view of a solid-core ring-magnet havinga cylindrical shape, further showing two cavities and a U-shaped vesselfor reaction mixture of magnetic beads and macromolecules. The ring ofcomplex between the magnetic beads and macromolecules just above the topof the magnet is shown.

FIG. 4D is a schematic of a side view of a standard ring magnet havingone full-length channel and a U-shaped vessel for reaction mixture ofmagnetic beads and macromolecules. The ring of complex between themagnetic beads and macromolecules just above the top of the magnet isshown.

FIG. 4E is a schematic of a perspective view of a of a solid-corering-magnet having a cylindrical shape, further showing two cavities anda V-shaped vessel for holding a reaction mixture of magnetic beads andmacromolecules shown in FIG. 4A. The ring or band of macromolecule/beadcomplex just above the top of the magnet is shown.

FIG. 4F is a schematic of a perspective view of a of a solid-corering-magnet having a cylindrical shape, further showing two cavities anda U-shaped vessel for holding a reaction mixture of magnetic beads andmacromolecules shown in FIG. 4B. The ring or band of macromolecule/beadcomplex just above the top of the magnet is shown and also a pipette isshown.

FIG. 5A is a schematic of a perspective view of a magnet plate havingmultiple solid-core ring-magnets that each has a cylindrical shape.

FIG. 5B is a schematic of a perspective view of a magnet plate havingmultiple solid-core ring-magnets that each has multiplecylindrical/conical-shaped cavities and an overall rectangular-prismshape.

FIG. 6 is a line plot chart of differences in pull forces measuredbetween a magnetic fixture and the solid core ring magnet (squares) orring magnet (triangles). The measurement was done using a digital forcegauge.

FIG. 7A is a line plot chart of the percent bead recovery over time from50 microliters of solution in a PCR plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7B is a line plot chart of the percent bead recovery over time from100 microliters of solution in a PCR plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7C is a line plot chart of the percent bead recovery over time from150 microliters of solution in a PCR plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7D is a line plot chart of the percent bead recovery over time from200 microliters of solution in a PCR plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7E is a line plot chart of the percent bead recovery over time from250 microliters of solution in a PCR plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7F is a line plot chart of the percent bead recovery over time from300 microliters of solution in a PCR plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7G is a line plot chart of the percent bead recovery over time from500 microliters of solution in a deep well plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7H is a line plot chart of the percent bead recovery over time from750 microliters of solution in a deep well plate, showing the differencebetween a standard ring magnet (triangles) and a solid-core ring-magnet(“X”) both having the same outer dimensions and magnetic grade.

FIG. 7I is a line plot chart of the percent bead recovery over time from1000 microliters of solution in a deep well plate, showing thedifference between a standard ring magnet (triangles) and a solid-corering-magnet (“X”) both having the same outer dimensions and magneticgrade.

FIG. 7J is a line plot chart of the percent bead recovery over time from2000 microliters of solution in a deep well plate, showing thedifference between a standard ring magnet (triangles) and a solid-corering-magnet (“X”) both having the same outer dimensions and magneticgrade.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

In many molecular biology procedures, macromolecules are needed in apurified form. For example, to prepare a DNA or RNA sample forsequencing, it needs to be extracted from any of a variety of clinicalsample types, such as tissue, blood, cheek swabs, sputum, forensicmaterial, FFPE samples etc. The initial extraction from the primarysample is followed by a multitude of enzymatic reactions called libraryconstruction. Each enzymatic reaction is followed by another extractionstep to isolate conditioned nucleic acid from the reaction mix. Theenzymatic reactions are typically followed by amplification (using PCR)and/or size selection (to limit the distribution of fragment sizes to anarrow band of a few hundred basepairs (e.g. 500-700 bp)). The workflowfrom primary sample to sequencing-ready DNA or RNA may involve from 5-10separate extraction steps. Throughout the workflow, the overall volumeof the mix containing the sample, as well as the sample container canvary significantly; typical volumes range from about 2000 μl to 35 μl.These workflows are often entirely automated to achieve the requiredprecision and throughput. The high degree of automation insequencing-related workflows has led to widespread adoption of magneticbead technology for extraction purposes, since alternative protocolsrequire either centrifugation or vacuum filtration, which are not easilyautomated.

Depending on the nature of the macromolecule to be extracted as well asthe matrix they are present in, magnetic beads (more precisely:paramagnetic beads) are coated with moieties (e.g., functional groups,other compounds) to which the macromolecules have affinity. For example,the beads might be coated with a carboxylic acid having moiety such assuccinic acid. The coupling between the beads and the macromoleculesmight also rely on streptavidin-biotin or carbo di-imide chemistry.Exemplary coatings include protein A, protein B, specific antibodies,particular fragments of specific antibodies, streptavidin, nickel, andglutathione. The beads themselves can vary in size, but will have anaverage diameter (e.g., 1 micro-meter). In some embodiments, theparamagnetic properties of the beads will result from integration ofiron into an otherwise non-magnetic substance (e.g., 4% agarose gel).Magnetic beads, as well as those that are already coated with variousaffinity groups, can be purchased from Sigma-Aldrich Corp. (St. Louis,Mo., USA), Life Technologies (Grand Island, N.Y., USA), ThermoScientific (Rockford, Ill., USA), EMD-Millipore (Billerica, Mass., USA),and New England Biolabs (Ipswich, Mass., USA).

In one application of the methods of the present invention, molecules(e.g., macromolecules) can be purified using magnetic beads byperforming the following steps:

-   -   a. mixing the magnetic beads having a particular        affinity-conferring coating with the molecule of interest in a        container (e.g., a vessel, an Eppendorf tube, a microplate well,        a deep well, a PCR well, round-bottom vessel);    -   b. after the mixing, allowing for specific binding between the        beads and the molecules, thus creating bead-molecule complexes;    -   c. placing the bottom of the vessel inside the cavity of a        solid-core ring-magnet;    -   d. allowing the bead-molecule complexes to aggregate (e.g.,        segregate) in a ring shape around the perimeter of the bottom of        the vessel (or of each vessel if using multiple ones); and    -   e. removing the supernatant, which would have unbound, undesired        molecules;    -   f. performing one or more wash steps by adding a suitable        solvent, e.g., ethanol, followed by removal of the same.        Additional steps can include re-suspending the bead-molecule        complexes in a solvent, so as to obtain a solution with a        desired volume and concentration. One can choose the appropriate        solvent so that the binding affinity between the beads and the        molecules is decreased, allowing them to dissociate from each        other. Or one can repeat the steps above to aggregate the        magnetic beads again to allow for additional separations,        depending on the buffer chosen.

Also the beads may be used to either bind the component of interest, forexample nucleic acid molecules, and during the method one discards thesupernatant and elutes the component of interest from the beads.Alternatively, one can let the beads bind to a component that one istrying to discard, leaving only the component of interest in thesupernatant. In this case, the supernatant is transferred to a new,clean vessel for use or further experimentation and the magnetic beadswith their unwanted molecules are discarded.

The above methods are generally automated using robotic systems (e.g.,automated liquid handling workstations) or aspirating/dispensingmanifolds. Usable workstations for automation include Agilent Bravo,Apricot Designs TPS-384, Beckman Biomk FX, Tecan Freedom EVO. The stepsof the present invention can be done manually e.g., using pipetting toremove/collect the supernatant.

Once a complex is formed between a macromolecule of interest and amagnetic bead (which might be formed via covalent as well asnon-covalent bonds), a magnetic field created by a magnet can beemployed to concentrate the bead-macromolecule complexes in a portion ofthe mixture (e.g., in a band in a solution). After that, the supernatantcan be aspirated (e.g., via pipetting) and the complexes are separatedfrom the mixture. Subsequently, the macromolecules can be separated fromthe beads, for example by eluting them via changes in the solution(e.g., buffer composition features such as pH and salt concentration).With currently known methods, this step results in large volumes ofeluted macromolecules. The present invention surprisingly allowsrecovery of an eluate that is of lower volume, of a higher yield, and ofa higher concentration. The process of recovery also is sped up with themagnet of the present invention.

The magnet of the present invention, in one embodiment is made from arare-earth metal such as neodymium. A neodymium magnet can have thechemical composition Nd₂Fe₁₄B, where Nd is neodymium, Fe is iron, and Bis boron. In some alternative embodiments, the magnet can also be madefrom samarium (e.g., sintered SmCo₅). The magnet can be covered with aprotective layer, for example a layer of nickel. Alternative coatingsinclude one or multiple layers, such as nickel, copper, zinc, tin,silver, gold, epoxy resin, or any other suitable material. Such coatingshelp, among other things, with preventing rusting of the iron component.In each of these embodiments, the full object is referred to as the“magnet”. The magnet can have a strength grade which for differentembodiments can be N35, N38, N40, N42, N45, N48, N50, or N52. Additionalmagnets with different grades, such as those with higher N-numbers(those that may be manufactured in the future) or different temperatureranges (H-grades), are also included among the embodiments of thepresent invention. The magnets (e.g., neodymium magnets) can be sinteredor bonded. Magnets can be purchased from K&J Magnetics, Inc., Jamison,Pa. For example, the cavities can be drilled into the magnet with adrill bit.

In one embodiment, shown in FIG. 1A, magnet 20 has two cavities, topcavity 8 and bottom cavity 10. Top cavity 8 descends from the center oftop surface 4, while bottom cavity 10 rises from bottom surface 6. Thesides of magnet 20 are surrounded by side wall 2. In the embodimentshown in FIG. 1A, both the magnet is cylindrical and a portion of thecavity wall is cylindrical-shaped. The cavities have walls that are inpart cylindrical-shaped and in part conical shaped. In an embodiment,the cavity wall can be any shape so long as a portion of the cavity wallhas a cylindrical shape to form a magnet field that attracts the beadsin a ring shape formation within the vessel. The term“cylindrical-shaped,” in this document, is used to refer tothree-dimensional structures that have sections that have ring-like(circular) outer boundaries. The term “cylindrical/conical-shaped,”refers to a cavity that has both features and in particular, hasthree-dimensional structures that have sections that have ring-like(circular) outer boundaries and a section of the base that is conical.The axes of the cylindrical sections, as defined, are parallel to theaxis of thickness (i.e., between the top surface and the bottom surfaceplanes) of the structure. Additionally, sections that are elliptical toa slight degree (e.g., the two radii differing by less than 5%) are alsoencompassed in the shape of the cavity. The cavity can be of any shapeso long as it can receive a vessel such that, when in use, the magneticfield causes the magnetic beads to form a ring within the vessel.

The overall structure, for magnet 20, is cylindrical when the presenceof cavities is ignored. In other words, the volume enclosed inside ofthe outside wall, bound above by the plane of the top surface (e.g., topplane), and bound below by the plane of the bottom surface (e.g., bottomplane) is cylinder-shaped. When referring to volumes, the terms topsurface and bottom surface are used to mean the plane of the top surfaceand the plane of the bottom surface, respectively.

For clarification, there are two pertinent volumes with respect to thecavities of the magnet of the present invention. The volume of thecavity itself, and the volume of solution in the vessel that, whenplaced into the magnet, resides generally within the cavity (i.e.,between the top plane and the lowest point of the cavity wall), or putanother way, from the lowest point of the cavity wall up to the beadring. In one embodiment, the volume of the cavity itself is betweenabout 5 and about 45 micro-liters (e.g., between about 5 and about 40,35, 30, 25, 20, 15 micro-liters. In another embodiment, the cavity has asize such that the volume of the solution in the vessel and that whichlies within the cavity up to the bead ring, in an embodiment, is betweenabout 5 and about 35 micro-liters (e.g., between about 5 and about 34,33, 32, 31, 30, 25, 20, 15, 10 micro-liters). The latter also refers tothe volume needed in the vessel to cover the macromolecule-bead ring soas to elute the beads from the macromolecules or to perform some otherexperiment. Note that a space exists between the cavity wall and thevessel placed within the cavity, and so a difference in volume existsbetween the cavity size and the volume of solution in the vessel andwithin the top plane. In the embodiment shown in FIG. 1A, two cavitiesare shown. However, one cavity can be used, in an embodiment, since itcreates a place for the vessel to be received. However, two cavities, inanother embodiment, are desired so that the magnets can be insertedduring assembly in either orientation, i.e., polarity. Accordingly, thepresent invention involves a magnet with one or more cavities (e.g.,two, three, four, five, six, seven, eight, nine, or ten, etc.).

In other embodiments, while the cavity wall has a portion that iscylindrical shaped, the overall magnet can be block-shaped, a bar, or aprism (e.g., rectangular-prism shaped). One such embodiment is shown inFIG. 1B. Shown are side wall 22, top surface 24, and top cavities (28A,28B . . . 28H) of magnet 40. Magnet 40 is generally a bar magnet withcurved ends and a number of cavities, whereas magnet 20 is a cylindricalmagnet with two cavities. With respect to the applications of themagnets, the focus is on the cavity as opposed to the full magnet.Therefore, both the cylindrical magnet (e.g., magnet 20) and the blockmagnet (e.g., magnet 40) are considered and referred to as solid coremagnets because regardless of the shape of the magnet that has cavities,the core is a solid filled magnet. “Solid core magnet” and “solid corering magnet” are used interchangeably herein. The word “ring” of thephrase “solid core ring magnet” connotes the shape of the top portion ofthe cavity wall or the ring of the paramagnetic bead/macromoleculecomplex that it forms. In other words, the term “solid core magnet” inthis document refers to magnets that have a solid core and a cavity wallwith at least a portion being ring shaped, regardless of whether themagnets are cylindrical shaped or rectangular-prism shaped.

FIG. 2A shows a top view of the magnet shown in FIG. 1A. Visible are topsurface 4, top cavity 8, as well as base surface 16 of the top cavity.From this figure, it is apparent that the cavity wall isring/conical-shaped. A cutout view as generated through the markings“3A” is shown in FIG. 3A.

FIG. 2B shows a top view of the magnet shown in FIG. 1B. Shown magnet 40is a block magnet, and has eight cavities. Shown in this figure are topsurface 24, top cavities (28A, 28B . . . 28H), and base surfaces (36A,36B . . . 36H) of the top cavities. Even though the outer boundary ofthis block magnet is shaped like a rectangular prism, this magnet isalso classified herein as a ring-magnet because the cavity walls arering/conical-shaped.

A cross section of the magnet previously introduced in FIG. 2A is shownin FIG. 3A. Magnet 20 shown in this figure has top cavity 8 and bottomcavity 10. The portion of top cavity 8 that descends from top surface 4toward the middle of the top cavity has a top ring shaped wall 12 and atop conical surface wall 16. The conical surface wall 16 is the portionof the cavity wall that has radii decreasing from that of the upperparts of the cavity wall to lower values until the cavity ends.Similarly shown are bottom cavity with bottom ring shaped wall 14 andbottom cavity conical surface 18. The shape of the cavity does not needto include a conical shape, and can be any shape (“V” shaped, “U” shapedor irregular shape) so long as it can receive the vessel, as describedherein. The top and bottom cavities, or their portions such as the wallsand surfaces, need not be the same as each other. However, having themthe same makes it easier to assemble them on a guide plate as well asmaking substitution of a magnet with another one easy. For embodimentsthat have identically shaped top and bottom cavities, a decision duringthe assembly of the magnets on a guide plate as to whether they have thesame or opposite polarity can be made by simply holding a random end ofeach of two magnets against each other. If they attract, they areoppositely polarized. If they repel, they share the same polarization.

A side view showing the long side of block magnet 40 is shown in FIG.3B. This figure shows side wall 22, top cavities (28A, 28B . . . 28H),and top cavity walls (32A, 32B . . . 32H). FIG. 3C shows the samemagnet, but from the viewpoint of the short side.

A comparison between a previously available magnet (referred to as a“standard ring magnet”) and the solid-core ring magnets of the presentinvention is shown in FIG. 4A through FIG. 4D. As should be immediatelyapparent, the standard ring magnet has a channel that runs through theentire thickness between the top and bottom ends of the magnet (FIG. 4Band FIG. 4D). In contrast, the solid-core ring magnet of the presentinvention, as the name implies, has a solid core and one or morecavities that do not create a channel/tunnel through the entirethickness of the magnet (FIG. 4A and FIG. 4C). Each of the cavitiesshown in FIG. 4A and FIG. 4C terminates with a conical surface. In thisembodiment, a conical surface allows accommodation of a vessel that hasa V-shaped bottom tip, whereas the diameter of the cavity above theconical surface allows accommodation of a vessel that has a U-shapedbottom tip. In striking contrast, while a standard magnet would lead toa high volume of sample being underneath the aligned level of themacromolecule, a solid-core ring magnet would allow a low volume ofsample being underneath the aligned level of the macromolecule/beadcomplex. Nucleic acid/bead band 62 aggregates at a lower position invessel 60 when using the solid core ring magnet of the present invention(See FIG. 4A), as compared to the position of the nucleic acid/bead band66 in vessels 64 using the standard ring magnet (See FIG. 4B). A lowerposition in the well is desirable since less elution buffer is generallyneeded to elute the DNA, leading to a higher DNA concentration.

The terms U-shaped vessel, vessel with a U-shaped bottom tip, and roundbottom shaped well are used interchangeable. The terms V-shaped vessel,vessel with a V-shaped bottom tip, and conical shaped well are also usedinterchangeably.

Overall, FIGS. 4A and 4C show conical shaped vessel 60 having a V-shapedbottom tip, nucleic acid/bead complex band 62, round shaped vessel 70,nucleic acid solution 72, and nucleic acid band 74. For comparison,FIGS. 4B and 4D show standard ring magnet 50 having standardchannel/tunnel 52, which is used for V-shaped vessel 64 to isolatenucleic acid 66, and standard ring magnet 54 having standard channel 56,which is used for U-shaped vessel 76 to isolate nucleic acid 80 fromsolution 78. As can be seen in the figures, the standard ring magnet ofFIG. 4B causes the nucleic acid/bead complex to sit higher in thevessel, as compared to the nucleic acid/bead complex shown in FIG. 4A.Accordingly, less elution buffer is needed when using the solid corering magnet of the present invention.

Additionally, FIGS. 4A and 4C show that the solid core ring magnet ofthe present invention is universal with respect to the type of vesselbeing used. It can be used with a “V” shaped vessels such as a PCR plateor a “U” shaped vessel such as a deep-well plate. Since either vesselshape can be used, the solid core magnet plate can be used to performseveral experiments or purification steps without having to switch toanother magnet plate having a different size/shaped magnet.

Even though the macromolecule is specifically a nucleic acid (e.g., DNA,RNA, PNA) in these figures, also included in other embodiments are othermacromolecules such as proteins (e.g., antibodies, peptides).Essentially, any macromolecule that can be made to adhere, reversibly ornot, to magnetic beads can be subjected to the methods disclosed herein.

Now turning to FIGS. 4E and 4F, the formation of the nucleic acid/beadcomplex can be seen. FIG. 4E is a cross-sectional view of FIG. 4A andFIG. 4F is a cross-sectional view of FIG. 4C. FIG. 4E shows theaggregation of the nucleic acid/bead band 62 and FIG. 4F shows theaggregation of nucleic acid/bead band 74. As can be seen from thisdrawing, the band forms a ring along the inner wall of vessels 60 or 70.The formation of the nucleic acid/bead band in a ring shape is afunction of the magnetic fields emitted by the solid core ring magnet,which are further described herein. Since a ring is formed along theinner vessel wall, pipetting the supernatant out (for example usingpipette 73), whether in an automated fashion or manually, is easilyperformed and allows one to leave the bead band in the vessel.

The location of the macromolecule ring band impacts the steps of themethodology for separating the macromolecules from the mixture. When thevessel is placed on the magnet, the magnetic beads in the solutionaggregate near the magnet at the place of the highest concentration ofthe magnetic field lines; this is where the magnetic field is generallythe strongest. Since the upper portion of the cavity wall is in theshape of a ring the beads form a ring in the bottom of the vessel, nearthe top of the magnet. After discarding the supernatant and washing theimmobilized beads with a wash solution, the next step is intended torecover the macromolecules from the beads. This is accomplished byexposing the beads to elution buffer, which will reverse the adherencebetween the macromolecules and the beads. The purified macromoleculesare then present in the elution buffer, which can subsequently beremoved from the vessel by aspiration. To effectively elute themacromolecules from the beads, one has to add enough elution buffer tocompletely cover the beads with buffer, so that effective elution cantake place. At the same time, one wants to keep the volume of elutionbuffer as small as possible so as not to dilute the macromoleculesunnecessarily. The volume needed is kept low because the magnet of thepresent invention is designed in such a way that the ring of beads willform as low as possible inside the vessel, regardless of the shape ofthe vessel.

Magnetic field lines are created by the magnets. The lines emanate fromone side of the magnet and terminate on the other. The direction of themagnetization is generally perpendicular to the surface(s) with thecavities, in other words, along the axis of the cavities. In particular,the magnets disclosed herein are magnetized through the thickness (i.e.,along the center axis running between the top surface plane and thebottom surface plane). Each cavity is surrounded by a top surface and abottom surface, and each such side (top surface and bottom surface) hasa certain polarity, which can be designated as north (N) or south (S).When the magnets having an overall cylindrical shape are assembled on aguide plate (an example of which is shown in FIG. 5A), they can bearranged in any number of arrangements including alternating rows,alternating columns, checkerboard arrangement or other pattern.Arrangements of polarities are embodied for any top plates that mighthave a different number of magnet receivers to accommodate various sizeplates (e.g., 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3ratio rectangular matrix).

Because the shape of the solid-core ring-magnet is different than thatof a standard ring-magnet with a channel/tunnel running through theentire thickness of the magnet, the magnetic field lines created aredifferent. In the solid-core ring magnet, the lines pass closer to thebody of the magnet and result in stronger pull forces because of theincreased amount of magnetic material. Experimental support for this isprovided in the exemplification section, Experiment 1 and in FIG. 6.Stronger pull forces facilitate quicker recovery of material, and alsofacilitate recovery of higher yields of material. See Experiment 2, FIG.7.

FIG. 5A shows magnet plate 90, within which there is top plate 82 (alsoreferred to as guide plate) that has 96 magnet receivers (i.e., theholes not shown in the figure, which receive the magnets). The magnetreceivers are arranged along 8 rows and 12 columns. Each magnet receiverreceives a magnet (e.g., 20A, 20B). Springs (98A, 98B, etc.) are placedaround shoulder posts (99B, etc.) at the corners of the top plate. Theshoulder posts, and the springs, pass through top plate 92 and baseplate 96. The springs allow flexibility in the leveling of the magnets,and thus any vessels placed in their cavities. With the springs,pipetting from the vessels can be accomplished more efficiently. In anembodiment, support plate 94 is a metal, and an affinity exists betweenthe support plate and the magnets. Further underneath, below both thetop plate and the support plate, is base plate 96. The top plate can befastened to the base plate by inserting shoulder posts (e.g., bolts)through the shoulder bolt receivers found at the corners of the twoplates. In some embodiments, the shoulder bolts and the springs can beon each of the four corners of the plates, whereas in other embodimentsthey can be in alternative locations (e.g., along portions of the edgesor on some of the corners only). The support plate is made from amaterial that has affinity to magnets. It can be from a metal such asiron, nickel, cobalt, or an alloy of different materials.

In a similar fashion to FIG. 5A, FIG. 5B shows a magnet plate. In thisembodiment, the magnets are block shaped. Similar elements, such as thethree plates (top, support, base), springs, and shoulder posts areusable with this embodiment. While not necessary, in the embodimentshown, all components except the magnets and the top plate are the sameas in FIG. 5A.

The integrated spring components enable complete liquid removal withouttip occlusion. The springs effectively cushion the wells, and allow theplates (e.g., top plate, support plate) to give way when tips (e.g.,pipette tips) come in contact with a well bottom. This compensates forphysical tolerances between labware and pipettors, each of which canotherwise compromise the precision of supernatant removal (e.g.,aspiration). In addition, in some embodiments the magnet plates aredesigned for automation; they have a standardized footprint to fit intostandard liquid handler plate nests, plate hotels, and stackers. Grippergrooves on the long sides provide space for robotic arms or gripperswhen moving microplates onto and off the magnet plates.

The solid-core ring-magnet, when used for isolating macromolecules,allows quicker recovery of the macromolecules, recovery of higherpercentages, and recovery of the macromolecules in smaller elutionvolumes. The solid core ring magnet, as described in the example,provides for better separation of the beads from the mixture. This isaccomplished because the solid core magnet provides additional forcethat is applied to the magnetic beads. In an embodiment, the solid coreprovides between about 1% and about 25% (e.g., about 20%, 15%, 10%, and5%) additional magnetic force, as compared to the standard ring magnet.See FIG. 6. The additional force provides for better, more efficientseparation. Accordingly, the magnet of the present invention has arecovery of the macromolecules between about 40% to about 99% (e.g.,about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%) recovery. Ascompared to a non-solid core magnet (e.g., a standard ring magnet), themagnet of the present invention improves recovery by about 1% to about60% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and55%).

Specifically, the magnet of the present invention is able to separatemore nucleic acid material and is able to do so faster and in fewercycles, as compared to the standard ring magnet. In an embodiment, themagnet of present invention is able to separate macromolecules that canadhere to magnetic beads in an amount that is 1× faster and up to 4.5times faster, as compared to a non-solid core magnet (e.g., a standardring magnet as shown in FIGS. 4B and 4D). Experimental support for theseimproved properties is provided in the exemplification section and inFIGS. 7A through 7J.

Standard conditions for forming the macromolecule-bead complex are knownin the art and can be found, for example, in Rohland, et al.,Cost-Effective High-Throughput DNA Sequencing Libraries For MultiplexedTarget Capture, Genome Research 22:939-946 and Supplemental Notes (theentire teachings of which are incorporated herein by reference). Forexample, reagent kits that can be used to form the macromolecule-beadcomplex are commercially available, such as the AMPURE composition fromBeckman Coulter, or such reagents can be made. One example of a solidphase reversible immobilization reagent that can be made and used withthe present invention is a MagNA composition, which is made from:

-   -   0.1% carboxyl-modified Sera-Mag Magnetic Speed-beads (FisherSci,        cat.#: 09-981-123)    -   18% PEG-8000 (w/v) (e.g. Sigma Aldrich, cat.#: 89510)    -   1M NaCl    -   10 mM Tris-HCl, ph 8.0    -   1 mM EDTA, pH 8.0    -   Optional: 0.05% Tween 20        To form the macromolecule-bead complex, in one embodiment,        0.5×-3× MagNA in an amount ranging from 10 microliters to 400        microliters can be added to the mixture.

EXEMPLIFICATION Introduction

Magnetic-bead-based nucleic acid purification is a standard technique inhigh-throughput sequencing. Purification steps occur at various pointsin the sample preparation workflow, from the original extraction of DNAout of a biological sample, to enzymatic conditioning steps, PCRcleanup, and size selection. To enable automated processing, the samplesare usually transferred from a primary container, like a collectiontube, Eppendorf vial or the like, to a microplate. Microplates exist inmany different specialized formats from 6 wells (2×3) to severalthousand wells. The most common format is the 96-well plate, wherein thewells, i.e. the individual cavities holding the samples, are arranged inan 8×12 array. Aside from the number of wells, microplates can varygreatly with regard to the volume per well, the shape of the wells, thematerials used, and other parameters depending on the intendedapplication. Despite all their differences, industry groups have agreedto a set of parameters defining certain dimensions of microplates withthe goal of maintaining their suitability for automated processing instandard robotic lab instruments. These standards are maintained by theSociety for Lab Automation and Screening (SLAS) and can be downloadedfrom their website atwww.slas.org/resources/information/industry-standards. The basicprinciple of magnetic bead separations includes the sequestration ofmagnetic beads from the reaction matrix by exposing them to a magneticfield. The magnetic force then immobilizes the beads, allowingsupernatant to be removed while the beads, with their attached payload,are retained.

The most common way of applying a magnetic field is achieved by placingthe microplate on top of a magnet plate that complements the microplate.Magnet plates are arrangements of permanent magnets in an array similarto the array of wells of the microplate types for which they are made.Just like there are various microplate types—with 24 wells, 96, 384 andso on, there are different magnet plates as well. Some magnet plates usepost magnets, where one post magnet is located in the center of 4 wells;also available are plates with bar magnets, where each bar magnet servesan entire row or column of wells of a microplate. A type of magnet plateis a ring magnet plate with 96 ring-shaped permanent magnets. The ringshape cavity is particularly useful because it produces a ring-shapedmagnetic field, causing the magnetic beads to aggregate in the same ringshape in the bottom of the microplate well. In this process, an area inthe center of the ring remains bead-free, allowing a pipet tip to reachthe well bottom and aspirate all liquid without disturbing the magneticbeads.

With the microplate still on the magnet, the beads are allowed to drybefore elution buffer is added to release the DNA from the beads. It isimportant to note that the volume of elution buffer necessary to achievecomplete elution must be sufficient to cover the beads entirely; if abead does not come into contact with elution buffer, the DNA will stayon the bead. At the same time, it is desirable to keep the elutionvolume as low as possible so as not to unnecessarily dilute the product(e.g. the purified, eluted DNA).

The minimum elution volume is a function of the location of the beadring inside the well. Lower bead rings allow for smaller elutionvolumes. FIGS. 4A and 4B show how the position of the bead ring dependson the geometry of the well and the magnet. The PCR well in FIG. 4Benters the ring magnet significantly lower than the PCR well in FIG. 4A.In an embodiment as shown in FIG. 4B, the elution volume to cover thebeads is about 35 μl. This is especially problematic because PCR plates,which have a well volume of only about 150-200 μl, are sometimes usedfor low volume reactions with low amounts of DNA. Eluting small amountsof DNA in larger volumes of elution buffer may lead to unacceptably lowDNA concentrations.

Other possible approaches use adapters between the magnet plate (withring magnets sized for round bottom wells as in 4D) to support a PCRplate. While viable in individual cases; the significant disadvantage isthat the adapter relies on specific PCR plate geometries; in otherwords, it is not a universal solution but only works with certain PCRplate types.

On the contrary, the solid core ring magnet is universal and achieveslow elution volumes. The solid core ring magnet of the present inventionalso separates the macromolecule/magnet beads faster and with morerecovery, as compared to standard ring magnets. The followingexperiments were designed to demonstrate the application of the solidcore ring magnet.

To verify the expected gain in performance, two experiments wereconducted.

Experiment 1: Comparison of the Pull Force Between a Solid Core RingMagnet and a Standard Ring Magnet

A solid-core ring magnet and a standard ring-magnet were manufacturedwith the properties shown in Table 1.

TABLE 1 Magnet Properties Solid Core Ring Magnet Standard Ring MagnetOuter Diameter 8.6 mm 8.6 mm Inner Diameter 4.3 mm to a depth of 4.3 mmthrough 2.5 mm, on both sides Thickness (Height) 11.5 mm 11.5 mmMagnetic Grade N50, NdFeB N50, NdFeB Magnetization Through the ThicknessThrough the Thickness Volume of Magnetic 613.2855 mm³ 500.8373 mm³MaterialThe Solid Core Ring Magnet contains about 22.45% more magnetic materialthan the regular ring magnet with the same outer dimensions. In anembodiment, the solid core ring magnet of the present invention hasbetween about 10% to about 30% more magnetic material, as compared to astandard ring magnet.

After this, an experiment was performed to determine the differences inpull forces between the two magnets across different distances. The datawas generated using a model ES30 test stand equipped with a force gaugeModel M5-20 and a Mitutoyo travel gauge, model ESM001 (all Mark-10Corporation, 11 Dixon Avenue, Copiague, N.Y. 11726, US).

FIG. 6 shows the results for comparing pull forces between a magneticfixture on one side and a solid core ring magnet or a standard ringmagnet on the other side. Both of the magnets used were grade N50,NdFeB, 8.6 mm diameter, and 11.5 mm thick. The ring magnet had an innerdiameter of 4.3 mm. The solid core ring magnet had two cavities, one oneach side, with a diameter of 4.3 mm and a depth of 2.5 mm. Both magnetswere magnetized through the thickness (i.e., along the center axis). Asseen, for a certain distance value, especially for lower values ofdistances, the solid core ring magnet has a stronger pull force. Becauseboth magnets are equivalent (same outer dimensions and magnetic grade)except that the standard magnet is drilled through all the way, thestronger pull forces in the solid core magnet result from the shape ofthe magnet, specifically the additional magnetic material present in thecore of the solid core ring magnet.

FIG. 6 shows the pull force between the test magnet and the magneticfixture. The magnetic fixture was the same in both tests.

Results:

Table 2 shows selected data points with the difference in pull force as% change.

TABLE 2 Pull Force Comparison; Selected Data Points Standard Ring MagnetSolid Core Ring Magnet Travel Load Travel Load % [mm] [gF] [mm] [gF]Difference 35 2 35.05 2 0.0% 33.5 2 33.5 2 0.0% 32.08 2 32.08 2 0.0%28.46 2 28.44 2 0.0% 22.18 6 22.19 6 0.0% 22 6 21.97 6 0.0% 21.52 621.56 8 33.3% 21.34 8 21.38 8 0.0% 15.04 20 15.06 22 10.0% 13.52 2613.52 30 15.4% 12.71 30 12.71 34 13.3% 11.5 38 11.53 42 10.5% 10.57 4610.54 52 13.0% 9.49 60 9.52 66 10.0% 8.08 80 8.05 94 17.5% 6.99 108 6.96124 14.8% 5.58 154 5.55 180 16.9% 5.33 168 5.36 190 13.1% 5.03 182 5.06206 13.2% 3.84 264 3.86 300 13.6% 3.2 326 3.24 370 13.5% 1.99 520 1.97614 18.1% 1.85 548 1.84 656 19.7% 1.51 660 1.52 788 19.4% 1.11 814 1.12944 16.0% 1.03 846 1.03 1028 21.5% 0.86 930 0.87 1138 22.4% 0.59 11020.58 1376 24.9% 0.43 1240 0.44 1536 23.9% 0.3 1390 0.31 1642 18.1% 0.211510 0.22 1768 17.1% 0.14 1634 0.15 1870 14.4%Result:

A comparison of the pull force generated between a regular ring magnetD=8.6 mm, d=4.3 mm, and H=11.5 mm, and a solid core ring magnet ofequivalent dimensions and grade shows significant differences in therange from 0 to about 15 mm of distance. The greatest difference wasmeasured at 0.58 mm distance with 24.9%. (A difference reading of 33%shown near the top of the table, at about 21.5 mm of distance, isconsidered noise. The signal, i.e. the pull force measured, is low atthis point, and the reading is surrounded on both sides by values of0%.)

Experiment 2: Bead Separation Time Comparison

Additional experiments were performed to investigate the bead separationtimes for the different magnets.

As described herein, the detection method by which the present inventionwas compared to current plate based magnetic separation devices byspectrophotometry. In standard high-throughput NGS DNA sequencingworkflows, each enzymatic process step is followed by a cleanup stepwhere the DNA is selectively bound to iron cored beads through theaddition of 0.1% carboxyl-modified Sera-Mag Speed-beads, 20%polyethylene glycol (PEG), and 2.5 M NaCl buffer in a mix ratio of 1.8×beads and buffer to 1× sample. The mixture is placed in a magneticfield, which pulls the beads and bound DNA to the sides of the well sothat the reagents, washes and/or unwanted fragments can be removed as asupernatant. The percent of bound material captured and the time ittakes for this capture to occur is of paramount importance formaintaining quality and throughput levels. Here we attempt to quantifythis recovery metric without the need to test the efficiency of thecapture chemistry. This was accomplished by simulating a given reactionvolume at a set end point, by replacing enzymatic components with waterwhile keeping the total reaction volume at 1.8× bead/PEG/NaCl mix:1×sample. We do not expect that beads bound with DNA will movesignificantly different through the PEG/NaCl matrix than those unboundto DNA.

A Detailed Procedure for Bead Detection:

A large quantity of 1.8×0.1% carboxyl-modified Sera-Mag Speed-beads(Thermo-Fisher Scientific, Pittsburgh Pa., USA, Cat number 09-981-123),20% polyethylene glycol (PEG) (Sigma-Aldrich, St. Louis Mo., USA, Catnumber 89510-250G-F), 2.5 M NaCl (Sigma-Aldrich, St. Louis Mo., USA, Catnumber S6546-1L), 0.05% Tween-20 (Sigma-Aldrich, St. Louis Mo., USA, Catnumber P9416-50ML) and 1× water were premixed and set aside. Apredetermined amount of bead/water mix was arrayed in groups of threeper time point to either an Eppendorf twin.tec semi-skirted PCR plate(Eppendorf AG, Hamburg, Germany, Cat number 951020362) or a RK Riplatedeep-well plate (BioExpress, Kaysville Utah, USA, Cat number 850356).Reaction volumes between 50-300 ul utilized the Eppendorf twin.tec plateand 500-2000 ul utilized the RK Riplate. Samples were arrayed in columnsso that three samples were used for every end-point and all samples hada zero time point used as a control. End-points for 50-100 ul trialswere 30 seconds-3 mins sampled in 30 second intervals, for 150-200 ultrials 30 seconds-5 min in 30 second intervals, for 200-750 ul trials 1min-5 min in 30 second intervals, and for 1000-2000 ul trials 2.5-25mins in 2.5 min intervals. Samples were arrayed using a 20-200 ul LTSmultichannel pipette (Rainin Instruments LLC, Oakland Calif., USA, catnumber L12-20XLS) or a 1000 ul single channel pipette (Gilson Inc.,Middleton Wis., USA, cat number P1000). After arraying, the samples wereleft on the bench for exactly 5 minutes to simulate DNA binding time.The 96-well plate was then placed on the magnetic separator plate and atimer was started. At the set end-point all liquid was removed from theend point wells using a multichannel pipette with a smooth constantpipetting motion so as to cause as little disturbance to the formed beadring as possible. Liquid was completely transferred to the correspondingwells of a second 96 well plate. All remaining time points of the samevolume were processed in a similar manner. Transferred samples were thenmixed 10× with a multichannel pipette to make sure any beads that mayhave settled had been completely resuspended. 50 ul, taken from themiddle of transferred sample, was then aliquoted to the correspondingwell of a 96 well flat bottomed plate (Thermo-Fisher Scientific,Pittsburgh Pa., USA, Cat number 12-565-501) for analysis.

Detection and Analysis Methods:

Samples and blanks were analyzed for absorbance based on publishedspecifications using a Tecan Infinite 200 Pro Multiplate reader withi-control microplate reader analysis software (Tecan Group, Ltd,Männedorf, Switzerland) measuring absorbance at 560 nm. Samples wereshaken in orbital mode at 3.5 amplitude for 3 seconds and then read at25 flashes per well. All plates were read in duplicate and the resultingabsorbance was averaged. Absorbance data was further analyzed using JMP11.2 software (SAS, Cary N.C., USA) for consistency between data points.Absorbance readings obtained for the blank wells were averaged togetherand used as a normalization control for all wells containing sample.Total percent of beads captured was calculated as a reverse function ofthe normalized absorbance of beads remaining in solution divided by thetotal absorbance of beads present in the control, or zero, time point.Results were then plotted in Excel (Microsoft Corp, Redmond Wash., USA)against the results of similar volume points obtained using othermagnetic separation devices.

TABLE 3 Std. Solid Core Std. Solid Core 50 ul Time Ring Mag. Ring Mag. %diff 100 Time Ring Mag. Ring Mag. % diff 30 80.07667297 94.69176 15.43%30 37.23995636 70.81196 47.41% 1 95.43984145 98.15866 2.77% 174.19305795 94.99906 21.90% 1.5 95.93133422 98.35559 2.46% 1.592.06909531 98.67371 6.69% 2 98.13500867 98.81871 0.69% 2 96.4728697999.03945 2.59% 2.5 98.34947811 99.6389 1.29% 2.5 97.81330423 98.887961.09% 3 98.28871191 99.40518 1.12% 3 98.69262933 99.60428 0.92% Std.Solid Core Std. Solid Core 150 Time Ring Mag. Ring Mag. % diff 200 TimeRing Mag. Ring Mag. % diff 30 26.07026337 50.14476 48.01% 30 19.3931103940.41279 52.01% 1 58.09414013 87.10766 33.31% 1 40.63989389 79.7691849.05% 1.5 74.4081237 96.27048 22.71% 1.5 51.16319869 93.50042 45.28% 291.05810939 98.5482 7.60% 2 71.00878141 96.39816 26.34% 2.5 94.2823015299.46902 5.21% 2.5 82.93686286 97.83296 15.23% 3 94.93285923 99.674614.76% 3 89.07426608 98.66614 9.72% 3.5 97.76385719 99.67894 1.92% 3.592.52722613 98.97777 6.52% 4 96.78087165 99.58156 2.81% 4 94.0142145899.10329 5.14% 5 98.59671404 99.77849 1.18% 5 92.9525902 99.53503 6.61%Std. Solid Core Std. Solid Core 250 Time Ring Mag. Ring Mag. % diff 300Time Ring Mag. Ring Mag. % diff 30 40.27648591 69.44254 42.00% 3031.69413085 65.15978 51.36% 1 59.90045115 89.61199 33.16% 1 48.7015659784.5415 42.39% 1.5 68.49710442 95.17374 28.03% 1.5 57.02298399 93.0529238.72% 2 80.3501216 96.40295 16.65% 2 66.49896343 95.38798 30.29% 2.587.17382752 98.30087 11.32% 2.5 76.29664731 97.13225 21.45% 390.64823436 98.90033 8.34% 3 82.53413641 97.63 15.46% 3.5 92.4569277799.30069 6.89% 3.5 85.17211198 98.85488 13.84% 4 95.00196603 99.653434.67% 4 89.16481993 99.01394 9.95% 5 95.23430771 99.67508 4.46% 592.34254341 99.29203 7.00% Std. Solid Core Std. Solid Core 500 Time RingMag. Ring Mag. % diff 750 Time Ring Mag. Ring Mag. % diff 30 34.5284275478.66054 56.10% 30 26.77121484 48.41286 44.70% 1 55.21212412 91.8377739.88% 1 41.5258199 81.40246 48.99% 1.5 68.91535241 94.73334 27.25% 1.552.50342389 90.49603 41.98% 2 78.2893862 96.90827 19.21% 2 60.7156417191.49368 33.64% 2.5 85.40605401 97.99248 12.84% 2.5 67.3773034 93.2595927.75% 3 87.73410062 98.62873 11.05% 3 72.559085 95.53839 24.05% 3.590.52157227 98.6125 8.20% 3.5 77.22843141 97.18311 20.53% 4 92.3239314998.51512 6.28% 4 84.04470492 94.31459 10.89% 5 93.04399126 99.042076.06% 5 85.71453813 97.83018 12.38% Std. Solid Core Std. Solid Core 1000Time Ring Mag. Ring Mag. % diff 2000 Time Ring Mag. Ring Mag. % diff 2.547.65286868 86.54932 44.94% 2.5 34.71005201 63.42803 45.28% 583.80165204 97.45749 14.01% 5 59.59467814 86.48007 31.09% 7.591.55886375 98.35018 6.91% 7.5 77.52805007 93.11305 16.74% 1092.38052749 98.53521 6.25% 10 79.11083052 94.19294 16.01% 12.595.53023817 98.59689 3.11% 12.5 83.89522614 95.7143 12.35% 1596.76273362 98.42809 1.69% 15 87.78547741 94.21133 6.82% 17.597.02336867 98.00825 1.00% 17.5 89.66750789 96.04108 6.64% 2097.28400404 99.08057 1.81% 20 87.96574837 97.4867 9.77% 22.5 96.5860318999.85315 3.27% 22.5 90.59770681 96.22719 5.85% 25 97.83177987 99.658381.83% 25 91.97137297 98.01907 6.17%

FIG. 7A shows the results for a 50 μL PCR plate, FIG. 7B for a 100 μLPCR plate, FIG. 7C for a 150 μL PCR plate, FIG. 7D for a 200 μL PCRplate, FIG. 7E for a 250 μL PCR plate, FIG. 7F for a 300 μL PCR plate,FIG. 7G for a 500 μL deep-well plate, FIG. 7H for a 750 μL deep-wellplate, FIG. 7I for a 1000 μL deep-well plate, and FIG. 7J for a 2000 μLdeep-well plate. In each of these results, especially for shorterattempted recovery times, it is clear that the percentage yield of therecovered beads is higher for a solid-core ring-magnet as compared to anequivalent ring-magnet. Similarly, when comparing similar amounts ofrecovery, it is clear that the solid-core ring-magnet allows recovery ofa similar percentage within a shorter period of time.

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A magnet for use in isolating macromolecules froma mixture in a vessel when the macromolecules adhere to paramagneticbeads to form a complex, wherein the magnet comprises: a. a solid corehaving an inner mass of the magnet, a first end and a second end; b. afirst surface at or near the first end of the magnet; c. a secondsurface at or near the second end of the magnet; d. one or more cavitiesextending into the solid core at or near the first surface, the secondsurface, or both; wherein the one or more cavities each have a cavitywall and a portion of the cavity wall forms a ring shape; e. at leastone side wall, wherein the side wall is in communication with the firstsurface and the second surface.
 2. The magnet of claim 1, wherein amagnet volume enclosed between the first surface, the second surface,and the side wall forms a cylinder.
 3. The magnet of claim 1, wherein amagnet volume enclosed between the first surface, the second surface,and further comprising side walls that form a rectangular prism.
 4. Themagnet of claim 1, wherein the cavity wall surrounds the cavity betweenthe first surface and at least a portion of the inner core, or betweenthe second surface and at least a portion of the inner core.
 5. Themagnet of claim 1, wherein the cavity wall comprises a base surface,wherein the base surface covers portions of the cavity not covered bythe portion of the wall having a ring-shape.
 6. The magnet of claim 5,wherein the base surface forms a conical shape, a “U” shape, or anirregular shape.
 7. A system for use in isolating macromolecules from amixture in a vessel when the macromolecules adhere to paramagnetic beadsto form a complex, wherein the system comprises: a. a magnet thatcomprises: i. a solid core forming an inner mass of the magnet, a firstend and a second end; ii. a first surface at or near the first end ofthe magnet; iii. a second surface at or near the second end of themagnet; iv. one or more cavities extending into the solid core at ornear the first surface, the second surface, or both; wherein the one ormore cavities each have a cavity wall and a portion of the cavity wallforms a ring shape; and v. at least one side wall, wherein the side wallis in communication with the first surface and the second surface; andb. the vessel for holding the mixture having the complex, wherein thevessel is placed on the magnet or is shaped to fit within the one ormore cavities.
 8. The system of claim 7, wherein a magnet volumeenclosed between the first surface, the second surface, and the sidewall of the magnet forms a cylinder.
 9. The system of claim 7, wherein amagnet volume enclosed between the first surface, the second surface,and further comprising side walls that form a rectangular prism.
 10. Thesystem of claim 7, wherein the one or more cavities each comprise aportion of the cavity wall that is conically shaped, “U” shaped, orirregularly shaped.
 11. A kit for use in isolating macromolecules from amixture in a vessel when the macromolecules adhere to paramagnetic beadsto form a complex, wherein the kit comprises: a. a magnet thatcomprises: i. a solid core forming an inner mass of the magnet, a firstend and a second end; ii. a first surface at or near the first end ofthe magnet; iii. a second surface at or near the second end of themagnet; iv. one or more cavities extending into the solid core at ornear the first surface, the second surface, or both; wherein the one ormore cavities each have a cavity wall and a portion of the cavity wallforms a ring shape; and v. at least one side wall, wherein the side wallis in communication with the first surface and the second surface; andb. the vessel for holding the mixture having the macromolecule, whereinthe vessel is placed on the magnet or is shaped to fit within the one ormore cavities.
 12. The kit of claim 11, wherein the kit furthercomprises magnetic beads.
 13. The kit of claim 11, wherein the kitfurther comprises one or more buffer compositions.
 14. A magnet for usein isolating macromolecules from a mixture in a vessel when themacromolecules adhere to paramagnetic beads to form a complex, whereinthe magnet comprises: a. a solid core having an inner mass of themagnet, a first end and a second end; b. a first surface at or near thefirst end of the magnet; c. a second surface at or near the second endof the magnet; d. one or more cavities at or near the first surface, thesecond surface, or both; wherein the one or more cavities each have atleast a portion of a cavity wall that has a shape configured to form amagnetic field within a vessel when the vessel in use and is placed atthe first surface or the second surface; e. at least one side wall,wherein the side wall is in communication with the first surface and thesecond surface.
 15. The magnet of claim 14, wherein a magnet volumeenclosed between the first surface, the second surface, and the sidewall forms a cylinder.
 16. The magnet of claim 14, wherein a magnetvolume enclosed between the first surface, the second surface, andfurther comprising side walls that form a rectangular prism.
 17. Themagnet of claim 14, wherein the cavity wall surrounds the cavity betweenthe first surface and at least a portion of the inner core, or betweenthe second surface and at least a portion of the inner core.
 18. Themagnet of claim 14, wherein the cavity wall comprises a base surface.19. The magnet of claim 18, wherein the base surface forms a conicalshape, a “U” shape, or an irregular shape.